Expression and characterization of human factor IX and factor IX-factor X chimeras in mouse C127 cells.
Human blood clotting factor IX, and two chimeric molecules of factor IX, in which the first epidermal growth factor-like domain or both epidermal growth factor-like domains have been replaced by that of human factor X, have been expressed in mouse C127 cells. The recombinants have been purified using a metal ion-dependent monoclonal antibody specific for residues 1-42 of human factor IX. All recombinant molecules are activated normally by human factor XIa in the presence of calcium ion. Activation of the factor IX recombinants by factor VIIa-tissue factor appears to be normal for the epidermal growth factor-1 exchange but considerably reduced for the construction containing both epidermal growth factor-like domains of factor X. The analysis of gamma-carboxyglutamic acid residues reveals that all of the purified recombinants are almost fully carboxylated. The extent of aspartic acid hydroxylation at residue 64 is 60% for all recombinants. The chimeric molecule with both epidermal growth factor-like domains from factor X has about 4% normal activity in the activated partial thromboplastin time assay. In contrast, the construct containing the first epidermal growth factor-like domain of factor X shows essentially normal clotting activity. Thus, it is unlikely that this domain is involved in a unique interaction with factor VIII.
Lin
S W
SW
Department of Biology, University of North Carolina, Chapel Hill 27514.
Smith
K J
KJ
Welsch
D
D
Stafford
D W
DW
D015055:1-Carboxyglutamic Acid / Q000032:analysis; D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000378:metabolism; D001483:Base Sequence; D001777:Blood Coagulation; D002118:Calcium / Q000494:pharmacology; D002460:Cell Line; D004815:Epidermal Growth Factor / Q000235:genetics*; D005164:Factor IX / Q000235:genetics* / Q000502:physiology; D015942:Factor VIIa / Q000494:pharmacology; D005170:Factor X / Q000235:genetics* / Q000502:physiology; D015945:Factor XIa / Q000494:pharmacology; D015870:Gene Expression; D006801:Humans; D006900:Hydroxylation; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics* / Q000502:physiology; D013925:Thromboplastin / Q000494:pharmacology
D011994:Recombinant Proteins; D001224:Aspartic Acid; D015055:1-Carboxyglutamic Acid; D004815:Epidermal Growth Factor; D005164:Factor IX; D005170:Factor X; D013925:Thromboplastin; D015942:Factor VIIa; D015945:Factor XIa; D002118:Calcium